Anti-inflammatory peptides and treatment to inhibit vascular leakage in injured tissues

ABSTRACT

Relatively small, synthetic anti-inflammatory peptides are provided having the primary sequence 
     
         T.sub.N --A.sub.1 --A.sub.2 
    
     This invention was made with Government support under Grant No. DA-00091 awarded by the National Institutes of Health. The Government has certain rights in this invention.

This invention was made with Government support under Grant No. DA-00091awarded by the National Institutes of Health. The Government has certainrights in this invention.

FIELD OF THE INVENTION

This invention generally relates to anti-inflammatory agents and to usesin reducing inflammatory responses, such as edema in connection withbrain, skin, mucosal and musculature injuries, and more particularly tothe use of relatively small, synthetic peptides that have the propertyof inhibiting vascular leakage.

BACKGROUND OF THE INVENTION

Inflammation is signaled by redness, swelling, heat and pain as areaction of the body against injury or assault. A variety of chemicalshave been implicated as chemical mediators of the inflammatory reaction,including histamine, kinins, prostaglandins, platelet-activatingfactors, leukotrienes, and, from nerve endings, substance P. Mediatorsof the acute inflammatory reaction seem to play roles in one or more ofincreasing vascular permeability, attracting leukocytes, producing pain,local edema and necrosis.

A variety of physiologic responses occur from the biological events thatconstitute the inflammatory processes. For example, Pinckard et al. atChapter 10 describe platelet-activating factors ("PAF") in the textInflammation: Basic Principles and Clinical Correlates (Gallin et al.Ed. 1988) This family of structurally related compounds appear topromote a variety of physiologic actions that are directly or indirectlyrelated to inflammatory reactions. The authors note that PAF has beenimplicated in the pathogenesis of human disease conditions such asendotoxin shock and organ transplantation rejection.

Swelling is a characteristic inflammatory response of tissues to injury.Swelling is produced by leakage of water and solutes of the blooddirectly into the tissue matrix. The increased leakiness of bloodvessels after injury may be due to direct damage of blood vessels or mayoccur after the release of substances such as histamine (inflammatorymediators) that open up gaps between endothelial cells that line theblood vessels. A mild degree of swelling (or edema) does not affect thefunctional integrity of injured tissues (except perhaps in the brain),but, in severe injuries, massive swelling distorts tissue architecture,impedes the delivery of oxygen to cells, and causes extensive fluid lossfrom the vascular compartment. Thus, a pharmacological agent capable ofinhibiting the swelling process may have therapeutic value in thetreatment of tissue injuries.

Inflammation is also involved in various chronic conditions, such asasthma, although it is not presently clear which inflammatory cells orwhich particular mediators are significantly involved in asthma.Persson, "The Role of Microvascular Permeability in the Pathogenesis ofAsthma", European Journal of Respiratory Diseases, Supp. No. 144, Vol.68, pp. 190-204 (1986), concludes that extravasated plasma protein isalways present in airways lumen of asthmatic subjects.

There are steroid and non-steroid, anti-inflammatory drugs known to theart. U.S. Pat. No. 4,579,844, inventors Rovee et al., issued Apr. 1,1986, discloses topically treating an inflammatory condition of the skinby use of the prostaglandin synthetase inhibitor concurrently with acorticosteroid. U.S. Pat. No. 4,404,198, inventor Kelley, issued Sep.13, 1983, discloses the topical application of a composition includingphenyl salicylate to treat inflammation. U.S. Pat. No. 3,980,778,inventors Ayer et al., issued Sep. 14, 1976, discloses a steroid for usein the topical, oral or parenteral treatment of skin and mucous membraneinflammations. Ibuprofen (a known anti-inflammatory agent) has beentested in connection with UV-B-induced inflammation, but was found tohave limited usefulness in treating sunburn reaction and is onlysomewhat more effective than placebo for the relief of symptomsassociated with UV-B-induced inflammation after high dose UV-Bphototherapy for psoriasis. Stern et al., Arch. Derm., 121, pp. 508-512(1985).

U.S. Pat. No. 4,801,612, inventor Wei, issued Jan. 31, 1989, disclosesthe use of inhibiting an inflammatory response in the skin or mucosalmembranes of a patient by administering corticotropin-releasing factoror its analogs.

The first corticotropin-releasing factor (CRF, also called CRH orcorticoliberin) to be characterized was a 41-residue peptide isolatedfrom ovine hypothalami by Vale et al. (1981). Subsequently, the sequenceof human-CRF was deduced from cDNA studies and shown to be identical torat-CRF. More recently, caprine, bovine, porcine, and white sucker fishCRF have been characterized. The CRF of hoofed animals show considerabledifferences from man, but the pig and fish sequences differ from thehuman/rat sequence by only 2 out of 41 residues.

For some mysterious reason, peptides with homologous structures tomammalian CRF are found in cells of certain frog skins and in theurophysis of fish. In fact, the structure of sauvagine, the 40 aminoacid peptide isolated from the skins of Phyllomedusa frogs, was reportedseveral years before Vale's description of ovine-CRF. The structure ofsucker fish urotensin I was reported just months after the descriptionof ovine-CRF and resulted from an independent line of inquiry byLederis's group in Canada. Although sauvagine and urotensin I releaseadrenocorticotropin from the pituitary, the functions of these peptidesin the tree-frog (Phyllomedusa species that live in arid regions ofSouth America) and in the sucker fish remain unknown. Recently, it hasbeen shown that the sucker fish has its own hypothalamic CRF which isvery close in structure to h/rCRF. Thus, the sucker fish would notrequire urotensin I for neuroendocrine regulation because it already hasCRF in it hypothalamus.

Rat corticotropin-releasing factor (hereinafter "CRF") is described inU.S. Pat. No. 4,489,163, inventors Rivier et al., issued Dec. 18, 1984.The amino acid sequence of both human and rat CRF is illustrated below:##STR1##

U.S. Pat. No. 4,415,558, inventors Vale, Jr. et al., issued Nov. 15,1983, describes the synthesis of sheep CRF, analogs, and isolation ofthe oCRF from ovine hypothalamic extracts. The synthetic oCRF was foundto lower blood pressure. The amino acid sequence of ovine (sheep) CRF isillustrated below: ##STR2##

The generally similar peptide, sauvagine, was described in RegulatoryPeptides 2, 1-13 (1981). Sauvagine is reported to have biologicalactivity in lowering blood pressure in mammals and stimulating thesecretion of ACTH and β-endorphin. The amino acid sequence of sauvagineis illustrated below: ##STR3##

U.S. Pat. No. 4,528,189, inventors Lederis et al., issued Jul. 9, 1985,and U.S. Pat. No. 4,533,654, inventors Lederis et al., issued Aug. 6,1985, describe white sucker and carp urotensin I, respectively, asstimulating ACTH and lowering blood pressure. The amino acid sequence ofcarp urotensin I is illustrated below: ##STR4##

The other CRF-related peptide, white sucker urotensin I, has an aminoacid sequence the same as the carp urotensin, except the amino acid atthe 24 position is isoleucine and the amino acid at the 27 position isglutamic acid.

Ling et al., BBRC, Vol. 122, pp. 1218-1224 (1984), disclose thestructure of goat CRF, which is the same as that for sheep CRF. Esch etal., BBRC, Vol. 122, pp. 899-905 (1984), disclose the structure ofbovine CRF which differs from sheep and goat CRF only by one amino acidresidue (number 33 which is Asparagine rather than the number 33 Serineof goat and sheep CRF). Porcine CRF has been isolated and characterizedby Patthy et al., Proc. Natl. Acad. Sci., Vol. 82, pp. 8762-8766 (1985).Porcine CRF shares a common amino acid sequence (residues 1-39) withrat/human CRF and differs from these only in position 40 and 41. Residue40 can be either asparagine or isoleucine and residue 41 isphenylalanine-amide.

These related peptides are summarized below (where the amino acids ofthe primary structure are illustrated by the IUPAC one-letter symbol).

    ______________________________________                                        Peptides of the Corticoliberin Superfamily                                    ______________________________________                                        CRF      SEEPPISLDL TFHLLREVLE MARAEQLAQQ                                     (human/rat)                                                                            AHSNRKLMEII*                                                         CRF      SEEPPISLDL TFHLLREVLE MARAEQLAQQ                                     (porcine)                                                                              AHSNRKLMENF*                                                         CRF      SEEPPISLDL TFHLLREVLE MARAEQLAQQ                                     (fish)   AHSNRKMMEIF*                                                         CRF      SQEPPISLDL TFHLLREVLE MTKADQLAQQ                                     (sheep/goat)                                                                           AHSNRKLLDIA*                                                         CRF      SQEPPISLDL TFHLLREVLE MTKADQLAQQ                                     (cow)    AHNNRKLLDIA*                                                         uro I    NDDPPISIDL TFHLLRNMIE MARIENEREQ                                     (sucker fish)                                                                          AGLNRKYLDEV*                                                         uro I    NDDPPISIDL TFHLLRNMIE                                                (carp)   MARNENQREQ AGLNRKYLDEV*                                              sauv.    pEGPPISIDLS LELLRKMIEI EKQEKEKQQA                                             ANNRLLLDTI*                                                          ______________________________________                                         *represents the amidation at the Cterminus                               

Both ovine and human/rat CRF have been used in clinical studies on theendocrine function of the pituitary-adrenal axis. Usually, doses of 1 to5 μg/kg have been injected intravenously to elicit endogenous release ofadrenocorticotropin and increases in plasma corticosteroids. Higherdoses of 10 μg/kg and 30 μg/kg of ovine-CRF were used by Orth et al."Effect of synthetic ovine corticotropin-releasing factor. Dose-responseof plasma adrenocorticotropin and cortisol.", J. Clin. Invest, 71 pp.587-595 (1983) in the initial assessment of this hormone in man. Thenon-endocrine effects of this hormone include symptoms such as flushing,shortness of breath and physical signs such as an increase in minutevolume, tachycardia (+20%) and possible hypotension. These parametersreturn to baseline levels within 30 min. and were not considered to beclinically harmful. The relative safety of CRF peptides is illustratedby the fact that CRF has been evaluated in normal children (aged 6-15years) at a dose of 1 μg/kg administered as an intravenous bolus, asreported by J. L. Ross, et al., "Ovine corticotropin-releasing hormonestimulation test in normal children", J. Clin. Endocrinol. Metab., 62,pp. 390-392 (1986).

However, it would be advantageous to have a peptide shorter than eitherCRF, sauvagine or urotensin I that is efficacious for reducing vascularleakage. For example, the costs of producing a peptide with seven totwelve amino acid residues would be much less than the costs ofproducing one that is forty or forty-one residues long because eachresidue must be added to the next residue in a step-wise fashion. Also,the possibilities of obtaining more selective biological actions ororal/topical activity from shorter peptides are potential advantages tobe considered.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide relatively small,synthetic peptides that can be used to inhibit inflammation of the skin,the mucosal membranes, and to decrease the leakage of blood componentsinto the brain tissue and musculature. Skin and mucosal membraneinflammations can occur from thermal (extremes of heat or cold) orradiation injury, or from noxious endogenous or exogenous substances.Leakage of blood components into the brain tissue, a condition calledvasogenic edema of the brain, can be produced by various adverse medicalconditions, such as brain ischemia, brain infarction, intracranialhemorrhage from neurosurgical operations, and so forth.

In one aspect of the present invention, an anti-inflammatory peptide isprovided having the primary sequence T_(N) --A₁ --A₂ --A₃ --A₄ --A₅ --A₆--T_(C) in which T_(N) is an amino terminal portion having a molecularweight less than about 600 daltons, A₁ through A₆ each is an amino acid(synthetic or natural) and together A₁ through A₆ constitute an activecore effective in providing anti-inflammatory activity to the peptide,and T_(C) is part of or comprises an amidated carboxyl terminal portion.

T_(N) is selected or modified to convey resistance against enzymaticdegradation of the active core. Each of A₁, A₂ and A₅ is a polar aminoacid in the D- or L-configuration and together A₁, A₂ and A₅ constitutea hydrophilic region for the secondary structure of the peptide. Each ofA₃, A₄ and A₆ is a nonpolar amino acid in the D- or L-configuration andtogether A₃, A₄ and A₆ constitute a hydrophobic region for the secondarystructure of the peptide. T_(C) is an amino group or an amidated aminoacid.

The class of anti-inflammatory peptides having the above-describedprimary sequence are sometimes hereinafter called "nocifensins" becausethey defend tissue against noxious stimuli and can be used asanti-inflammatory agents.

A therapeutically effective amount of an inventive nocifensin peptidecan be administered, preferably by intravenous, intradermal orsubcutaneous means, such as in doses from about 1 to about 200 μg/kg.Such administrations reduce the permeability of brain and centralnervous system blood vessels and are of therapeutic value in thetreatment of tissue injury, such as brain and central nervous injuries.Administrations also provide clinical benefits when used to limit orminimize leakage of blood constituent into tissue during surgery, toalleviate pain and discomfort, and to prevent further swelling forpatients already experiencing inflammation.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 illustrates a spaced-filling model of a synthetic peptideembodiment for the invention where the model is being viewed from theN-terminus down the axis, or backbone, towards the C-terminus; and,

FIG. 2 plots the data from a bioassay conducted with the prior arthuman/rat CRF and four inventive peptide embodiments, respectively, andillustrates vascular leakage inhibition in a dose related manner(horizontal axis) with respect to saline controls (vertical axis).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

When the brain is injured such as in brain ischemia, or infarction, thenvasogenic edema occurs and the increased amounts of water compress anddistort brain tissue architecture and impede the delivery of oxygen tobrain cells. The patient can lose consciousness and stop breathing. Whenthe skin or a mucosal surface is injured by extreme heat or cold, byradiation or assaulted by noxious substances, it is converted from acondition of balanced fluid exchange to a state in which serum and itssolutes freely pass into the surrounding tissues. In general, substancesare released, either from nerve endings or from cells within the injuredtissue, that produce increased vascular permeability. Fluids andproteins in the blood then move from the vascular compartment to thetissue compartment with pain, swelling and tissue damage as a result.

In the present invention, a nocifensive peptide (or a salt form thereof)can be used to inhibit inflammation of a mammal's skin, mucosalmembranes, or where there have been (or will be) lacerations of themusculature or injury to the brain. Administration can includeintravascular, oral or topical means at dosages of between about 0.001to 5 mg/kg body weight. However, administration is preferably byintravenous, intradermal, or subcutaneous injection with at least asingle dose being given, preferably from about 1 to about 200 μg/kg bodyweight, and can be about two hours before deliberate lacerations of themusculature (such as during abdominal or orthopedic surgery) and can beup to three days after surgery or accidental injury. Administration ismost preferably via the blood stream, but local injections into thecerebrospinal fluid, brain, or into the muscle can be used.

Because the nocifensive peptides have the property of inhibitingvascular leakage from tissues, they are useful in a number of differenttherapeutic applications. Specific tissues for which clinical usage ofthese peptides may be applied include skin and mucosal membranes(eyelids, nasal membranes, oropharyngeal membranes, upper respiratorytract, esophagus, lower digestive tract), skeletal muscle, smoothmuscle, cardiac muscle, blood vessels of the brain, and blood vessels ofthe lungs and kidneys. For example, therapeutic uses of these peptidesinclude administration to treat thermal burns, irradiation burns,frostbite, or for other inflammatory conditions of the skin. Thepeptides may be used to reduce swelling, pain, and plasma extravasation.For irritants deposited on the upper airways or in chronic allergicconditions such as asthma, the peptides may be used to decreaseirritancy, bronchial inflammation, edema and plasma extravasation. Forlacerative or traumatic injuries to all tissues, such as might occurafter knife wounds, surgical operations, and automobile accidents, thepeptides may be used to reduce swelling, pain, and inflammation. Fortissue infarcts, which result in tissue hypoxia, ischemic anoxia andedema, such as occur after brain strokes or myocardial infarcts, thepeptides may be used to reduce passage of blood constituents into thetissue matrix and enhance survival of the remaining tissues. Forpreventing the actions of endogenous or exogenous chemicals thatdirectly injure the endothelium, such as endotoxins or inflammatorymediators, resulting in the clinical condition of septic shock, thepeptides may be useful in reducing the loss of blood volume.Administration may be used for preserving the integrity of the vasculartree of an organ prior to or during its removal for transplantation,such organs being kidneys, liver, or heart.

The nocifensive peptides may be administered in combination with apharmaceutically acceptable carrier, such as isotonic saline, phosphatebuffer solution or the like. Topical administration is also feasiblesince the peptides are relatively small.

Compositions and excipients useful for the administration of smallpeptides through the nasal mucosa are, for example, described by U.K.patent application No. 8719248, published Feb. 24, 1988, applicantSandoz Limited. Topical compositions with enhanced penetration throughintact skin preferably include a potentiator, many of which aredescribed by U.S. Pat. No. 4,091,090, issued May 23, 1978, inventorSipos. The nocifensive peptides form pharmaceutically acceptable saltswith organic and inorganic acids and can be administered in salt form.Examples of suitable acids for the formation of pharmaceuticallyacceptable salts are hydrochloric, sulfuric, phosphoric, acetic,benzoic, citric, malonic, salicylic, malic, fumaric, succinic, tartaric,lactic, gluconic, ascorbic, maleic, benzenesulfonic, methane- andethanesulfonic, hydroxymethane- and hydroxyethane- sulfonic.

Salts may also be formed with suitable organic pharmaceuticallyacceptable base addition salts. These organic bases form a class whoselimits are readily understood by those skilled in the art. Merely forpurposes of illustration, the class may be said to include mono-, di-,and trialkyamines, such as methylamine, dimethylamine, andtriethylamine; mono-, di- or trihydroxyalkylamines such as mono-, di-and triethanolamine; amino acids such as arginine, and lysine;guanidine; N-methylglucosamine; N-methylglucamine; L-glutamine;N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine;tris(hydroxymethyl)aminomethane; and the like. (See for example,"Pharmaceutical Salts," J. Pharm. Sci. 66 (1): 1-19 (1977).

Nocifensive peptides of the invention have the primary sequence T_(N)--A₁ --A₂ --A₃ --A₄ --A₅ --A₆ --T_(C). Thus, the amino terminal, orT_(N), has a molecular weight less than about 600 daltons and isselected or modified to convey resistance against enzymatic degradationof the active core for the inventive peptide. Preferred moieties forT_(N) are di-, tri-, tetra-, or penta-peptides or derivatives thereof.Particularly preferred for T_(N) are D-asparagine, D-tyrosine orD-glutamine. Particularly preferred derivatives for T_(N) areN-methylphenylalanine or pyroglutamic acid. Other examples of moietiesuseful to convey resistance against enzymatic degradation arep-chloro-or p-nitro-phenylalanine, lower alkyl alkylated arginine andlysine, and the o-ethyl ethers of asparagine and glutamine. Unnaturalamino acids to stabilize or to increase potency and prolong duration ofaction are illustrated, for example by the article of Nestor et al.,Jour. of Medic. Chem., 31 (1), pp. 65-72 (1988).

A₁ and A₂ are each a basic polar amino acid (synthetic or natural).Thus, A₁ and A₂ can be in the D- or L- configuration and each ispreferably selected from arginine and lysine.

Each of A₃, A₄ and A₆ is a basic non-polar amino acid preferablyselected from leucine, isoleucine and methionine, while A₅ is an acidicpolar amino acid, preferably glutamic acid and aspartic acid.

The six amino acid residues, A₁ through A₆, together constitute anactive core for the peptide that is effective in providinganti-inflammatory activity to the peptide. Since each of A₁, A₂, and A₅is a polar amino acid (in the D- or L- configuration), together A₁, A₂,and A₅ constitute a hydrophilic region for the secondary structure ofthe peptide. Since each of A₃ , A₄, and A₆, is a nonpolar amino acid (inthe D- or L-configuration), together A₃, A₄, and A₆ constitute ahydrophobic region for the secondary structure of the peptide. When theactive core is a mixture of D- and L-configurations, then they arebelieved to form a "random coil". However, when all are, for example, inthe L- configuration, then they are believed to form an α-helix. Ineither event, because the three polar residues will associate together,as will the three hydrophobic residues, the molecules have anamphiphilic character.

Turning to FIG. 1, a space-filling model of a particularly preferredembodiment is illustrated where the red hatching represents polarresidues while the blue hatching represents hydrophobic residues. Themodel is viewed from the N-terminus down the axis of the molecule.Reading clockwise in the hydrophilic region, A₁ illustrated as arginine,A₅ as glutamic acid, and A₂ as lysine. Reading counter-clockwise in thehydrophobic region, we see illustrated A₄ as methionine, A₃ as leucine,and A₆ as isoleucine. Thus, the particularly preferred peptideembodiment being illustrated has the primary structure:

    T.sub.N --R K L M E I--T.sub.C

where

T_(C) is D-leucineamide and

T_(N) is no particular A.A.

FIG. 1 also illustrates that the hydrophobic and the hydrophilic regionsof the inventive peptides each radially outwardly extends from abackbone of the active core with the hydrophilic region and thehydrophobic region being generally opposed. That is, the polar residuesdo not interdigitate with the hydrophobic residues.

It is believed possible to vary the just-described residues bysubstitution of either the second basic residue (that is, A₂), theacidic residue (A₅) or the third hydrophobic residue (A₆) with D- orL-asparagine or glutamine, although potency is a likely to bediminished. However, replacement of the acidic residue with asparagineor glutamine may increase the stability of the molecule because theacidic carboxyl ligand would no longer be available to react with thebasic amino ligand of the arginine or lysine residues.

Finally, T_(C) is an amino group or an amidated amino acid. A preferredamidated amino acid is D-leucine amide because this residue is resistantto enzymatic degradation from the C-terminus. A non-polar aromaticresidue such as phenylalanine is also a suitable moiety for T_(C).

The nocifensin peptides can be synthesized by various suitable chemicalmethods, preferably by solid phase synthesis, manual or automated, asfirst developed by R. B. Merrifield and described by J. M. Stewart andJ. D. Young in "Solid Phase Peptide Synthesis" (1984). Chemicalsynthesis joins the amino acids in the predetermined sequence startingat the C-terminus. Basic solid phase methods require coupling theC-terminal protected alpha-amino acid to a suitable insoluble resinsupport. The described nocifensin peptides require benzyhydrylamine(BHA) or 4-methylbenzyhydrylamine (MBHA) resins to produce theC-terminal amide upon completion. Amino acids for synthesis requireprotection on the alpha-amino group to ensure proper peptide bondformation with the preceding residue (or resin support). Followingcompletion of the condensation reaction at the carboxyl end, thealpha-amino protecting group is removed to allow the addition of thenext residue. Several classes of alpha-protecting groups have beendescribed, see J. M. Stewart and J. D. Young in "Solid Phase PeptideSynthesis" (1984), with the acid labile, urethane-basedtertiary-butyloxycarbonyl (Boc) being the historically preferred. Otherprotecting groups, and the related chemical strategies, may be used,including the base labile 9-fluorenylmethyloxycarbonyl (FMOC). Also, thereactive amino acid side-chain functional groups require blocking untilthe synthesis is completed. The complex array of functional blockinggroups, along with strategies and limitations to their use, have beenreviewed by M. Bodansky in "Peptide Synthesis" (1976), and, J. M.Stewart and J. D. Young in "Solid Phase Peptide Synthesis" (1984).

Solid phase synthesis is initiated by the coupling of the describedC-terminal alpha-protected amino acid residue. Coupling requiresactivating agents, such as dicyclohexycarbodiimide (DCC) with or without1-hydroxybenzo-triazole (HOBT), diisopropylcarbodiimide (DIIPC), orethyldimethylaminopropylcarbodiimide (EDC). After coupling theC-terminal residue, the alpha-amino protecting group is removed bytrifluoroacetic acid (25% or greater) in dichloromethane in the case ofacid labile tertiary-butyloxycarbonyl (Boc) groups. A neutralizing stepwith triethylamine (10%) in dichloromethane recovers the free amine(versus the salt). After the C-terminal residue is added to the resin,the cycle of deprotection, neutralization and coupling, withintermediate wash steps, is repeated in order to extend the protectedpeptide chain. Each protected amino acid is introduced in excess (threeto five fold) with equimolar amounts of coupling reagent in suitablesolvent. Finally, after the completely blocked peptide is assembled onthe resin support, reagents are applied to cleave the peptide from theresin and to remove the side chain blocking groups. Anhydrous hydrogenfluoride (HF) cleaves the acid labile tertiary-butyloxycarbonyl (Boc)chemistry groups. Several nucleophilic scavengers, such asdimethylsulfide and anisole, are included to avoid side reactionsespecially on side chain functional groups.

A number of the inventive nocifensin peptides have been synthesized andtested by means of a bioassay for anti-inflammatory activity. Thesynthesis of a particularly preferred one of these peptides isspecifically described by Example 1. Example 2 then describes thebioassay procedure used with Table 1 summarizing a number of thesepeptides and Table 2 showing comparative dose response data.

EXAMPLE 1

The synthesis of an inventive nocifensin peptide having the formula:

    D-Ala.His.Ser.D-Asn.Arg.Lys.Leu.Met.Glu.Ile.D-Leu-NH.sub.2

was conducted sequentially from the C-terminal amide end on a4-methylbenzhydrylamine hydrochloride resin (MBHA HCl resin), ascommercially available with amine substitution range of 0.4 to 0.6millimoles per gram resin (CalBiochem, Inc., Bachem, Inc.). The amountof resin equal to one millimole of active amine was washed withappropriate solvents (dichloromethane and/or methanol). Neutralizationof the MBHA resin with triethylamine (TEA) in dichloromethane (DCM)removed the salt form, enabling sequential addition of thetertiary-butyloxycarbonyl (Boc) protected amino acid derivatives. TheC-terminal residue, Boc-leucine (monohydrate), was coupled to thereactive resin amine groups with one molar dicyclohexycarbodiimide (DCC)in dichloromethane (DCM). Generally equimolar amounts of coupling agent(DCC) and Boc-amino acid are added in excess (five-fold) relative to theresin. The Boc-amino acids are dissolved in dichloromethane (DCM) and/ordimethylformamide (DMF), depending on the particular residue'ssolubility.

After acidic deprotection with trifluoroacetic acid solution (25% indichloromethane) and neutralization with triethylamine (10%) indichloromethane, the stepwise building continued toward the amino end.Boc-arginine (Tos), Boc-histidine (Tos) and Boc-D-asparagine werecoupled in a 9:1 mixture of dimethylformamide (DMF) and dichloromethane(DCM). P-toluenesulfonyl (Tos) groups are used to protect the guanidinoof arginine and the imidazole of histidine. A xanthyl (Xan) ring wasused to protect the amido group of asparagine. 2-Chlorobenzyloxycarbonyl(2-Clz) was used for the lysine side chain. The benzyl ether (Bzl) wasused with the hydroxyl of serine, and the benzyl ester (OBzl) used forblocking the carboxyl of glutamic acid.

Finishing the protected amino acid couplings, gave the followingintermediate: Boc-D-Ala.His(Tos).Ser(Bzl).D-Asn(Xan).Arg(Tos).Lys(2Clz).Leu.Met.Glu(OBzl).Ile.D-Leu-resinsupport. The final cleavage and deprotection of the protected peptideresin required acidolysis with hydrogen fluoride (HF), the nucleophilicscavengers dimethylsulfide and anisole (or equivalent) and was done at0° C. for 30 minutes. Preferably, cleavages at a preliminary lowertemperature, 0° C. for 20 minutes, followed by 0° C. for 30 minutes todecrease the probability of side reactions. Alternately, a "low HF"procedure requiring higher concentrations of dimethyl sulfide relativeto hydrogen fluoride (3:1 by volume) at 0° C. for three hours mayprecede normal HF cleavage with MBHA and BHA resins to avoid sidereactions.

Removal of the HF under vacuum in the cleavage apparatus precedesmultiple washes of the peptide-resin with dry ethyl ether and/orchloroform for extraction. Filtration follows with one molar aqueousacetic acid, with the obtained filtrate frozen and lyophilized. The usedresin is weighed to determine the yield of peptide, and the need for anyre-extraction.

Example 1 illustrates the synthesis of a preferred embodiment(D-a-H-S-n-R-K-L-M-E-I-l-NH₂). A series of the nocifensive peptides havebeen similarly synthesized and tested. The approximate purity of thepeptides prepared ranged from about 57% to 94% (as determined by HPLC).Amino acid sequences and purity data, are set out in Table 1 for anumber of these inventive compounds.

The abbreviations used in the table on bioassay results and elsewhere inthis disclosure are: standard single letter codes for amino acids, andthe lower case of the single letter represents the D configuration ofthe amino acid. All peptides were synthesized with the carboxylamideterminus or other than carboxylic acid. By "pE" is meant pyroglutamyl,"M°" means methionine sulfoxide, and "Fm" means N-methylphenyalanine.

EXAMPLE 2

The peptides were synthesized by solid phase methods as described oranalogous to that described by Example 1, and the approximate purity ofmost was obtained by high-pressure liquid chromatography and is given inthe Table 1. The peptides were dissolved either in saline or an inertdextrin solvent (Molecusol) and injected intravenously into apentobarbital-anesthetized rat. The rat's hind paw was then immersed in58°-60° C. water for 1 min and the increase in paw weight, as an indexof edema and swelling, was measured 30 min. later. For some peptides,the ability of the substances to inhibit vascular leakage after muscleinjury was also tested. Normally, the paw weight of saline-treatedanimals (controls) will increase by about 68-81% after heat injury. Theincrease in weight is relative to the contralateral non-heated paw.

In screening the bioactivity of new peptides, a dose of 5 mg/kg i.v. wasinitially used. In later studies, when greater activity was expected, ascreening dose of 1 mg/kg i.v. was used. For peptides that exhibitedparticularly significant activity, a full dose-response analysis wasconducted according to the method of Litchfield and Wilcoxon.

Thus, each peptide was injected intravenously (at the amounts specified,usually 1 or 5 mg/kg, or by full dose-response analysis) 10 min beforeimmersion of the right paw in 58° C. water for 1 min, and weights ofboth paws were obtained 30 min later. To obtain statistical accuracy, aminimum of six animals were used in each group. The % wt increase wascalculated as (wt of heated paw/ wt of unheated paw) ×100 and convertedto % of the saline control group values, which were run concurrently.The weight of the paws of the saline-treated animals did not increaseafter immersion in room temperature (22° C.) water, but increased by 68to 81% after heat.

                                      TABLE 1                                     __________________________________________________________________________    Inventive Peptides                                                            (Primary Structure)                                                                              HPLC Peak %                                                                           % Saline Value                                     __________________________________________________________________________     ##STR5##              62 73                                                                              36 ± 4 52 ± 6                                                               (at 5.0 mg/kg) (at 5.0 mg/kg)                  ##STR6##              84 83 89 94                                                                        86 ± 4 49 ± 4 59 ± 4  34 ±                                            (at 5.0 mg/kg) (at 5.0 mg/kg) (at 5.0                                        mg/kg) (at 1.0 mg/kg)                           ##STR7##              na   89 ± 3                                                                         (at 5.0 mg/kg)                                 ##STR8##              na 87 92 87 88 na 86 88 na                                                         60 ± 7 52 ± 4 49 ± 4 46 ± 3 77                                   ± 4 53 ± 3 37 ± 5 36 ± 3 23 ±                                       (at 5.0 mg/kg) (at 1.0 mg/kg) (at 1.0                                        mg/kg) (at 1.0 mg/kg) (at 5.0 mg/kg) (at                                      5.0 mg/kg) (at 5.0 mg/kg) (at 5.0 mg/kg)                                      (at 5.0 mg/kg)                                  ##STR9##              79  83 80 81                                                                       36 ± 4 28 ± 5 25 ± 2 19 ±                                             (at 1.0 mg/kg) (at 1.6 mg/kg) (at 1.2                                        mg/kg) (at 0.4 mg/kg)                           ##STR10##             73 58 69 85 94 94                                                                  70 ± 2 47 ± 4 38 ± 6 24 ± 4 31                                   ± 5 24 ± 7                                                                   (at 1.0 mg/kg) (at 1.0 mg/kg) (at 1.0                                        mg/kg) (at 1.0 mg/kg) (at 1.0 mg/kg) (at                                      1.0 mg/kg)                                      ##STR11##             84 82 70                                                                           86 ± 3 35 ± 6 30 ± 5                                                     (at 1.0 mg/kg) (at 1.0 mg/kg) (at 1.0                                        mg/kg)                                         __________________________________________________________________________     lower case denotes the Damino acid                                            # means Peptide dissolved in 22% dextrin solvent (Molecusol)                  M.sup.o = methionine sulfoxide, F.sup.m = Nmethylphenylalanine, pE =          pyroglutamic acid                                                             (1), (2), (3), and (4) = note these four are further described in Table 2

Turning to the data of Table 1, we see that 29 different nocifensivepeptides were prepared and shown to provide a statistically significantinhibition of edema when administered to mammals whose paws were exposedto an inflammation causing situation. Many of these nocifensive peptidesprovided a remarkable diminution of swelling. Since "100%" under the "%Saline Value" of Table 1 means no diminution of swelling, then peptidessuch as that having the primary structure "a H S n R K L L E I l" (witha value of 19% saline value when administered at 0.4 mg/kg), "a H S n RK L M e I l" (at 24% saline value) and "a H S n R K I I E I l" (at 24%saline value), in addition to the others designated (1)-(3), areparticularly worthy of special note in substantially preventingswelling. The four inventive analogs indicated by the (1)-(4)designations were then further analyzed, as shown by the data of Table2, for bioactivity and can be compared to the known human/rat CRF.

                  TABLE 2                                                         ______________________________________                                        Peptide               ED50 (95%         Po-                                   # for                 Confidence)       ten-                                  FIG. 2                                                                              Primary Structure                                                                             Mg/kg i.v.  Slope cy                                    ______________________________________                                              h/rCRF          0.013 (.002-.089)                                                                         32.0                                        (1)   a H S n R K L M E I l                                                                         0.12 (.06-0.25)                                                                           3.6   1                                     (2)   a H S N R K L M E I l                                                                         0.56 (.28-1.13)                                                                           2.4   1/5                                   (3)   a H S n R R L M E I l                                                                         0.63 (.29-1.39)                                                                           4.1   1/5                                   (4)   a H S n R K L L E I l                                                                         0.72 (.36-1.44)                                                                           2.9   1/6                                   ______________________________________                                         Log-Probit analysis according to Litchfield and Wilcoxon (1949).         

Turning to Table 2 and FIG. 2, we see that the slope value of the priorknown human/rat CRF is greater than, and not parallel to, that of thenocifensin peptides. This means it is likely that human/rat CRF acts ina different manner from the nocifensins. The maximum inhibition ofswelling obtained with h/r CRF was 64%, whereas with the nocifensins itwas greater than 80%. Thus, the nocifensins appear to be considerablymore efficacious in reducing vascular leakage than the prior known h/rCRF.

In sum, the inventive nocifensins should find use in treating injuredtissues such as involved in stroke (brain tissue), burn or frostbiteinjury (skin tissue), asthma (mucosa), and surgery (abdominal and/ororthopedic muscles).

It is to be understood that while the invention has been described abovein conjunction with preferred specific embodiments, the description andexamples are intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims.

I claim:
 1. A peptide having the primary structure:

    T.sub.N --A.sub.1 --A.sub.2 --A.sub.3 --A.sub.4 --A.sub.5 --A.sub.6 --T.sub.C

where T_(N) is an amino terminal portion having a molecular weight lessthan about 600 daltons and is selected to convey resistance againstenzymatic degradation, T_(C) is D-leucineamide or phenylalanine, A₁ isD- or L- arginine or lysine, A₂ is D- or L- arginine or lysine, A₃ is D-or L- methionine, leucine or isoleucine, A₄ is D- or L- isoleucine,leucine, or methionine, A₅ is D- or L- glutamic acid, glutamine, oraspartic acid, and A₆ is D- or L- isoleucine, leucine, or asparagine. 2.The peptide as in claim 1 wherein:T_(N) is a di-, tri-, tetra- orpenta-peptide or a derivative thereof.
 3. The peptide as in claim 2wherein:T_(N) includes D-asparagine, D-tyrosine or D-glutamine adjacentto the amino acid at A₁.
 4. The peptide as in claim 2 wherein:thederivative includes N-methylphenylalanine or pyroglutamic acid.